The proposed research aims to study regulation of macromolecular transport through plant intercellular connections, the plasmodesmata (PD). PD interconnect most cells within a mature plant and are critical for maintaining and regulating communication within and between different plant tissues. To examine the mechanism(s) by which the control of PD transport occurs, we exploit plant viruses, pirates of plasmodesmata, that move between host cells through these channels. For widespread infection, plant viruses must move from the initially infected cell to its surrounding cells. Because PD are the only connections between adjoining plant cells, plant viruses use these channels as their major routes of passage from cell to cell. PD transport of Tobacco mosaic virus, one of the best studied plant viruses, occurs with the help of a single virally-coded factor, the movement protein (MP). MP, therefore, represents a powerful molecular tool to study macromolecular transport through PD. In the proposed research, we shall continue to utilize this experimental approach, focusing on one of the most intriguing, yet poorly understood, aspects of PD transport - its regulation. The need to tightly control PD transport is inherent in its central role during plant-virus interactions as well as during normal plant development and morphogenesis. The molecular mechanisms by which such PD transport control is achieved remain largely unknown. In the current project, we have isolated several plant factors that are involved in these regulatory pathways, likely functioning as "checkpoints" of distinct stages of PD transport. The planned experiments will continue and expand this research direction. Specifically, each of the following two aims of the proposed work will seek to study a different aspect of PD regulation, together contributing toward a single goal of understanding of the molecular mechanisms that control PD transport. I. Differential phosphorylation of MP as an "On/Off' switch of PD transport. We have identified an ER-associated protein kinase (ERPK) that specifically phosphorylates MP at the Ser-37 residue, activating its PD-gating activity. Earlier, we also identified a PD-associated protein kinase (PDPK) that phosphorylates MP at its Ser-258, Thr-261, and Ser-265 residues and acts as a negative regulator of the MP ability to gate PD. Thus, ERPK and PDPK represent regulatory "checkpoints" for MP transport through PD. Here, we shall further study the effects of these two enzymes on MP (e.g., recognition of and targeting to PD and alterations in the a-helical and protease-resistant domains of MP thought to be involved in its PD targeting and gating activities) and on developmental regulation of PD permeability, identify and initially characterize their cellular substrates, and use reverse genetics to determine the phenotypic effects of the ERPK and PDPK knockouts/knockdowns on PD transport of plant viruses and cellular proteins. II. Control of PD transport by the MP-glucanase and GrIP/pdGRP/glucanase systems. We showed that MP directly interacts with ?-1,3 glucanase, an enzyme that destroys callose located in the neck region of PD and known to restrict of PD transport. We hypothesize that the MP-glucanase interaction promotes relaxation of the callose sphincter, resulting in PD gating. On the other hand, we discovered a GrIP/pdGRP/glucanase system, in which a PD-associated glycine-rich protein (pdGRP) interacts with ?-1,3 glucanase, potentially inhibiting its activity and leading to tightening of the callose sphincter. The levels of pdGRP itself are modulated by its interacting protein, GrIP. Thus, modulation of the ?-1,3 glucanase by MP and cellular factors likely represents another regulatory "checkpoint" in the PD transport pathway. We shall examine the mechanisms by which MP-glucanase interaction and the GrIP/pdGRP/glucanase system control PD permeability via callose accumulation. We shall study the MP-glucanase and pdGRP-glucanase interactions and their effects on the enzymatic activity of ?-1,3 glucanase. We shall investigate how GrIP binding to pdGRP modulates accumulation of pdGRP, and explore the role of the GrIP/pdGRP/glucanase system in developmental regulation of PD permeability. [unreadable] [unreadable] [unreadable]